HUMAN GENE THERAPY 12:1663–1672 (September 1, 2001)Mary Ann Liebert, Inc.

In Vivo Persistence of Retrovirally Transduced Murine Long-Term Repopulating Cells Is Not Limited by

Expression of Foreign Gene Products in the Fully orMinimally Myeloablated Setting

ELIZABETH KANG,1,4 NEELAM GIRI,2,4 TONG WU,2 STEPHANIE SELLERS,2 MARTHA KIRBY,2

YUTAKA HANAZONO,3 JOHN TISDALE,1 and CYNTHIA E. DUNBAR2

ABSTRACT

Many nonmalignant hematologic disorders could potentially be treated by genetic correction of as few as5–10% of target lineage cells. However, immune system clearance of cells expressing gene products perceivedas foreign could be limiting. There is evidence that tolerance to foreign proteins can result when myeloabla-tive conditioning is used, but this limits the overall applicability of such techniques. Therefore, we sought toevaluate the engraftment of hematopoietic stem cells carrying a foreign transgene after low-dose irradiationby comparing in vivo survival of murine long-term repopulating cells (LTRC) transduced with either a retro-viral vector expressing the bacterial neomycin phosphotransferase gene (neo) or a vector containing neo genesequences but modified to prevent protein expression (nonexpression). First, marrow cells from congenicdonors were transduced with either vector and transplanted into recipients treated with standard dose irra-diation of 800 rads. High-level engraftment and gene marking resulted, without differences in the markinglevels or pattern of persistence of the cells between cells transduced with either vector. Low-dose irradiationat 100 rads was tested using higher cell doses. Marking levels as high as 10% overall were obtained, againwith no differences between mice receiving cells transduced with the neo versus the nonexpression vectors.To investigate a potentially more immunogenic protein, marrow cells were transduced with a vector con-taining the green fluorescent protein (GFP) gene, and their persistence was studied in recipient mice receiv-ing 100 rads. Stable GFP expression in 5–10% of circulating cells was observed long term. We conclude thateven with very low dose conditioning, engraftment by genetically modified LTRC cells at clinically significantlevels can be achieved without evidence for clearance of cells known to be expressing immunogenic proteins.

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INTRODUCTION

MANY NONMALIGNANT OR CONGENITAL DISORDERS, such asmetabolic and enzyme deficiency diseases, can be po-

tentially treated by the genetic correction of only a fraction(5–10%) of target lineage cells. However, there is concern overthe potential for host immunologic clearance of cells carryingforeign or new genes. In the murine model, muscle injection ofvectors expressing human erythropoietin resulted in anemia,due to an antibody to the foreign cytokine that cross-reactedwith native erythropoietin (Tripathy et al., 1996). Even in pa-

tients with a crippled immune system due to human immuno-deficiency virus (HIV) infection, cytotoxic T lymphocytes de-veloped against mature T cells transduced with a retroviral vec-tor carrying the herpes thymidine kinase gene, greatly limitingin vivo persistence of these cells (Riddell et al., 1996). Evencorrective genes not considered foreign to a normal host couldelicit an immune response in recipients lacking the endogenousgene product. Alpha-L-iduronidase (ID)-deficient dogs infusedwith transduced marrow cells without conditioning of the re-cipient developed both humoral and cytotoxic responses to thealpha ID enzyme (Lutzko et al., 1999).

1Molecular and Clinical Hematology Branch, National Institute of Diabetes and Digestive and Kidney Disorders, Bethesda, MD 20892.2Molecular Hematopoiesis Section, Hematology Branch, National Heart, Lung, and Blood Institute, Bethesda, MD 20892.3Division of Genetic Therapeutics, Center for Molecular Medicine, Jichi Medical School, Tochigi, Japan.4These authors contributed equally to this work.

However, persistence of genetically modified cells has beenachieved when the gene is introduced via the hematopoieticstem cell compartment after myeloablative conditioning. Weobserved that rhesus macaques infused with mature T lympho-cytes transduced with either a neomycin phosphotransferasegene (neo) or a nonexpression vector have long-term markingonly with cells containing the nonexpression vector. In contrast,equivalent levels and persistence of cells containing the neo andnonexpression vectors were observed following reinfusion oftransduced hematopoietic stem cells after myeloablative condi-tioning. Challenge of these animals with mature T cells trans-duced with a second neo expression vector resulted in persis-tence of the cells long term (Heim et al., 2000), suggesting thatimmunologic rejection can be avoided by introduction of theforeign gene via the hematopoietic stem cell compartment.

Recent improvements in gene transduction methods have re-sulted in stable marking levels of 5–15% or higher in both ca-nine and primate models, adequate for many potential clinicalapplications (Kiem et al., 1994, 1998; Tisdale et al., 1998).However, these studies were performed in the setting of fullymyeloablative radiation. To assess the role of myeloablation forboth the engraftment and prevention of immunologic rejectionof transduced cells, the in vivo survival of hematopoietic cellstransduced with a neo expression vector was compared to thesurvival of cells transduced with a vector containing neo genesequences, but modified to prevent translation (neo nonexpres-sion) using a murine hematopoietic stem cell (HSC) gene trans-fer model. To determine the maximal level of engraftment bycells transduced with either the neo or nonexpression vector,recipient mice were first conditioned with myeloablative radi-ation at a dose of 800 rads. Subsequently, a second cohort ofrecipient mice was conditioned and transplanted using very-low-dose irradiation at 100 rads. Finally, it is possible that someproteins such as the neo gene product although foreign, maynot be sufficiently immunogenic to result in stimulation of animmune based clearance of vector-containing cells. Thus a fi-nal cohort of mice was transplanted with cells transduced witha green fluorescent protein (GFP)–expressing vector after con-ditioning with 100 rads to assess the ability of low-dose irra-diation to allow engraftment and persistence of cells express-ing foreign proteins known to be highly immunogenic.

MATERIALS AND METHODS

Mice

Six- to eight-week-old C57BL/6J (C57) female donor miceexpressing the Ly 5.2 Ag were purchased from Jackson Labo-ratory (Bar Harbor, ME). Six- to ten-week-old female recipientF1 heterozygotes expressing both Ly 5.2 and 5.1 were producedby crossing C57Bl6 females (Ly 5.2) to congenic BL6/SJLmales (Ly 5.1).

Bone marrow harvesting

Donor mice were treated with recombinant human granulocytecolony-stimulating factor (rhGCSF) (200 mg/kg per day) (Am-gen, Thousand Oaks, CA) and recombinant rat stem cell factor(SCF) (50 mg/kg per day) (Amgen) by subcutaneous (s.c.) injec-

tion for 5 consecutive days. Fourteen days following the fifth doseof cytokines, bone marrow was harvested by flushing from thefemurs and tibiae, and then dispersed through a 21-gauge needle.Mononuclear cells (MNC) were isolated by density gradient cen-trifugation over lymphocyte separation media (LSM; OrganonTeknika, Durham, NC), washed twice in Dulbecco’s minimal essential medium (DMEM; Life Technologies/Gibco/BRL,Rockville, MD) supplemented with 10% fetal bovine serum (FBS;HyClone, Logan, UT), 4 mM L-glutamine, 50 mg/mL penicillinand streptomycin, and cultured on non-tissue culture-treated 100-mm culture dishes (Falcon, Franklin Lakes, NJ).

Vectors

The G1Ne ecotropic producer cell line is a GPE-86 packag-ing cell line clone releasing the G1Ne retroviral vector encod-ing the neomycin resistance gene expressed from the long ter-minal repeat (LTR) promoter at a titer of 5 3 106/ml (Yu et al.,1998). The PL-1e producer cell line was derived by infectingGPE-86 packaging cells with amphotropic G1PL-1 vector(Hanazono et al., 1999) supernatant, and isolating a high-titerclone by limiting dilution. Relative titer was estimated by RNAslot blot and by Southern blot comparison of 3T3 cells trans-duced with either G1Ne or PL-1E supernatants. The titer of thePL-1e producer clone was estimated to be 1 3 105/ml. TheGFP-expressing ecotropic MgirL22Y vector is described else-where and has a titer of approximately 5 3 106/ml (Persons etal., 1997).

Retroviral transduction

Marrow MNCs were transduced by exposure to filtered vec-tor supernatant collected from confluent producer cell linesgrown in DMEM with 15% FBS. All transductions were car-ried out in the presence of 10 ng/ml recombinant murine inter-leukin-3 (IL-3; R & D Systems, Minneapolis, MN), 100 ng/mlrecombinant rat stem cell factor (SCF; Amgen), 50 ng/ml re-combinant human IL-6 (Amgen), 100 ng/ml recombinant hu-man Flt-3 ligand (Immunex, Seattle, WA), and 5 mg/ml prota-mine sulfate (Sigma, St. Louis, MO). Transduction cultureswere continued for a total of 96 hr with daily replacement ofvector supernatant and cytokines. Cells were harvested by rins-ing plates vigorously with phosphate-buffered saline (PBS), fol-lowed by trypsinization of adherent cells. Cells were washedand resuspended in PBS with 0.1% bovine serum albumin(BSA; Life Technologies) to a final concentration of 80 3 106

cells/ml for injection.

Transplantation

Recipient mice received either 800 or 100 rads of total bodyirradiation (TBI) using a cesium source irradiator, (Gammacell,Nordian, Kanata, Canada) followed by tail vein injection of ei-ther 10 3 106 (800 rad recipients) or 120 3 106 (100 rad re-cipients) transduced cells given in a split dose on 4 consecu-tive days.

Sample collection and flow cytometric analysis

Peripheral blood was obtained by retroorbital collection fromrecipient mice at 2, 6, 10, 20, and 24 weeks post-transplanta-

KANG ET AL.1664

tion. Donor cell engraftment was assessed by mixing 25 ml ofblood with 75 ml of 5% BSA in PBS, and 2 ml each of anti-Ly5.2-FITC and anti-Ly 5.1-PE (Pharmingen, San Diego, CA) orappropriate isotype controls (Pharmingen). After 30 min atroom temperature, the samples were red cell lysed using the Q-prep procedure and apparatus and analyzed using the CoulterEpics XL-MCL Flow Cytometer (Beckman Coulter, Miami,FL). For GFP analysis, 25 ml of blood was processed identi-cally but without antibody staining.

PCR analysis

DNA was extracted from 200 ml of peripheral blood via phe-nol- chloroform extraction (Sambrook et al., 1989). PCR analy-ses for the presence of the neo expression vector (G1Ne) andnonexpression vector (PL-1e) were carried out using 100 ng oftemplate DNA using the following primers and conditions: 59-TCC ATC ATG GCT GAT GCA ATG CGG C-39 and 59-GATAGA AGG CGA TGC GCT GCG AAT CG-39 for the outerreaction run for 30 cycles, and 59-ATA CGC TTG ATC CGGCTA CCT G-39 and 59-GAT ACC GTA AAG CAC GAGGAA-39 for the inner reaction run for 20 cycles. Amplificationof actin sequences to normalize for amplifiable DNA contentwas performed as described (Wu et al., 2000). The Perkin-Elmer Cetus standard DNA PCR reagent kit with AmpliTaqDNA polymerase was used (Norwalk, CT), and Ci800 32P-la-beled dCTP (Amersham Pharmacia Biotech, Piscataway, NJ)was added during the inner PCR reaction. Both reaction cyclesconsisted of 95°C for 1 min, 54°C for 1 min, and 72°C for 2min. Products were separated by polyacrylamide geleletrophoresis (PAGE) with quantitation of band intensity us-ing a PhosphorImager (Molecular Dynamics, Sunnyvale CA)and comparison of test sample image intensity of G1Ne, PL-1e, or GFP amplification products/actin to a standard curve gen-erated from copy number controls amplified concurrently as de-scribed previously elsewhere. (Hanazono et al., 1999). Cyclenumbers were chosen to ensure linear amplification of the testsequence and the actin sequence in the range of marking beingquantified.

For the third experiment, PCR for the MgirL22Y GFP vec-tor was performed using the following primers and conditions:59-GCC ACA AGT TCA GCG TGT CC-39, and 59-AGCTC-GATGCGGTTCACCAG-3 9 at 95oC for 1 min, 54oC for 1 min,and 72oC for 2 min for 30 cycles. Quantitation was performedvia image analysis using Sybr green staining (Biowhittaker) anda Fuji camera (Stamford, CT), and comparison of test sampleimage intensity of PL-1e or GFP amplification products/actinto a standard curve generated from copy number controls am-plified concurrently.

Reverse transcriptase PCR

To assess for the presence of the neo transgene product, RNAfrom peripheral blood was isolated using RNA STAT-60 (Tel-Test, Friendswood, TX), and PCR was performed after prepar-ing cDNA from purified RNA samples treated with DNase byreverse transcriptase (Perkin-Elmer RNA PCR kit; Roche Mo-lecular Systems, Branchburg, NJ) using the following condi-tions and primers: 59-TCC ATC ATG GCT GAT GCA ATGCGG C-39 and 59-ATA CGC TTG ATC CGG CTA CCT G-39

for the outer reaction run at 95°C for 1 min, 55°C for 1 min,and 72°C for 2 min for 26 cycles, and 59-ATA CGC TTG ATCCGG CTA CCT G-39 and 59-GAT ACC GTA AAG CAC GAGGAA G-39 for the inner reaction run at 95°C for 1 min, 55°Cfor 1 min, and 72°C for 2 min for 30 cycles.

Statistical analyses

Significance testing for comparisons between groups wasperformed using a Wilcoxon Rank Sum test. Analysis for thetrend of GFP expression was performed by a one-sample Stu-dent’s t-test using the slopes generated from linear regressioncurves for each mouse analyzed at the different time points (Mi-crosoft, Redmond, WA).

RESULTS

Experimental design

In all experiments, a direct comparison was made betweenengraftment and persistence of murine bone marrow cells trans-duced with vectors expressing foreign genes known to be im-munogenic, and cells transduced with a “nonexpression” vec-tor containing cDNA sequences from the bacterialneo-resistance gene and the b-galactosidase gene, but mutatedto prevent actual expression of the gene products. We have pre-viously published details regarding the construction of the non-expression vector PL-1 and documented that no expression offull-length or truncated gene products could be detected byfunctional assays or Western blot analysis (Hanazono et al.,1999; Heim et al., 2000).

Because persistence of transduced cells was the variable be-ing studied, a model was required that would ensure that therewas no recipient immune response against determinants otherthan the transgene expressed in donor cells, but would also al-low quantitative assessment of donor cell engraftment in the re-cipients. CD45 antigens have been shown to be immunogenicin settings of low-dose irradiation (van Os et al., 2001). Thus,parental Ly 5.2 C57Bl6 mice were used as marrow donors, andtransduced cells were transplanted into Ly 5.1/Ly 5.2 het-erozygous F1 recipients (Fig. 1).

Comparison of expression versus nonexpression vectormarking in the myeloablated setting

In the first set of experiments, recipient animals received 800rads and were transplanted with a dose of 10 3 106 marrowmononuclear cells per mouse. We have previously shown thisdose of total body irradiation to be lethal in 100% of C57 micenot given bone marrow rescue (Giri et al., 2001). As expected,high-level and durable donor cell engraftment as assessed byflow cytometry of Ly 5.2 versus Ly 5.2/Ly 5.1 cells was ob-tained in groups of mice receiving either G1Ne (expression) orPL-1e (nonexpression) vector-transduced marrow cells.

Overall gene marking levels were similar between the micereceiving cells transduced with neo expression and nonexpres-sion vectors, and ranged from 30% to 50% (mean 40.5% 6 2.1SD) in the G1Ne group and 3-50% (mean 18.5% 6 16.8 SD)at the 6-month time point in the PL-1e group, assuming a sin-

LOW-DOSE TBI AND PERSISTENCE OF TRANSGENES 1665

gle vector copy per cell as assessed by PCR. The lower titer ofthe PL-1e producer clone may explain the resulting lower over-all marking level. There was no difference in the pattern ofmarking over time between the two groups, and no evidencetherefore for specific immune-mediated clearance of cells trans-duced with the neo expression vector. The level of vector-con-taining cells did not decline over time in either group and per-sisted beyond 6 months in both groups (Fig. 2).

Comparison of expression versus nonexpression vectormarking in the minimally radiated setting

To determine if these persistent high levels of marking fromthe expression vector could be achieved using very low-doseirradiation, the experiment was repeated using the same vec-tors; however, the recipients were irradiated with only 100 rads.This dose was chosen to allow easily quantifiable engraftmentand marking. Even very high cell doses of transduced cells havenot given quantifiable results without ablation (Schiffmann etal., 1995). To obtain adequate engraftment for analysis, the celldose was increased to 120 3 106 cells (Quesenberry et al.,1997). Engraftment ranged from 10-25% (mean 22 6 3.9) inthe G1Ne group and 10-25 %(16.3 6 4.2) in the PL-1e group

over the 6-month time period as shown in Fig. 3. In fact at the6-month time point, engraftment was significantly higher in theG1Ne group (p , 0.05, Wilcoxon Rank Sum test) showing nodetriment to engraftment by cells expressing the neomycin tran-script. Overall gene marking was similar between the twogroups of mice, ranging from 0.1% to 10% in the G1Ne groupand 0.1% to 3% in the PL-1e group (p . 0.05, Wilcoxon RankSum test). When corrected for the level of engraftment, genemarking in the G1Ne group ranged from 1% to 28% and in thePL-1e group from 1% to 13%. The level of gene marked cellsdid not decline over time and persisted beyond 6 months asshown in Fig. 3. Moreover, transcription of the neo expressionvector was confirmed by positive reverse transcriptase PCR ofperipheral blood RNA obtained from two animals approxi-mately one year post transplant as shown in Fig. 4.

Finally, we confirmed these findings with a vector express-ing the enhanced GFP. In this model, in vivo expression of theforeign gene can be easily monitored and the GFP gene hasbeen shown to be highly immunogenic in mice, including theC57BL6 strain (Stripecke et al., 1999). In this experiment, 100-rad conditioning was used and the MgirL22YGFP vector wascompared to the Pl-1e nonexpression vector (Persons et al.,1997). A total of 120 3 106 cells transduced with either the

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FIG. 1. Experimental design. Marrow from C57Bl6 mice expressing Ly 5.2 was harvested 14 days after cytokine prestimula-tion and transduced with either G1Ne (a neo-expressing vector) or Pl-1e (a nonexpressing vector) in experiments 1 and 2, andin experiment 3 with either Pl-1e or MGirL22Y (GFP-expressing vector), by four daily supernatant exposures in cytokine-sup-plemented suspension culture. The recipient mice, an F1 hybrid of C57Bl6 and SLJ expressing both Ly 5.1 and Ly 5.2 and thusdistinguishable from the donor by flow cytometry, were conditioned with a single dose of irradiation at 800 rads (experiment 1)or 100 rads (experiments 2 and 3). Each animal then received either 10 million (experiment 1) or 120 million (experiments 2 and3) transduced cells by tail vein injection.

GFP vector or the PL-1e vector were infused into each mouse.Again there was no detriment to engraftment by vector-ex-pressing cells as donor cell engraftment was significantly higherin the GFP group at the 6-month time point (p , 0.05, WilcoxonRank Sum test).

Overall engraftment ranged from 9.6% to 25.1% (mean16.6 6 7.5) in the Pl-1e group and 28.3% to 47.1% (mean31.5 6 16.8%) in the GFP group, and persisted beyond sixmonths as determined by PCR and shown in Fig. 5. Overall,gene marking ranged from 0.1% to 25.2% in the PL-1e recip-ients and from 7 to 54.7% in the GFP recipients as measuredby PCR (Fig. 5). We measured the percentage of cells ex-pressing GFP by flow cytometry, and these correlated well withthe results obtained by PCR analysis for the GFP gene (Fig. 6).The level of marking long-term was in fact significantly better(p , 0.05, Wilcoxon Rank Sum test) with the GFP-expressingvector, and there was a significant trend toward an increasingnumber of cells expressing GFP as well as in the number ofGFP vector-containing cells over time (p , 0.05 Student’s t-test), suggesting that no active immune rejection was occurring(Figs. 5 and 6).

DISCUSSION

Hematopoietic stem cell gene therapy has long been pursuedas a definitive treatment for many congenital and acquired dis-orders; however, most applications will require high-level en-graftment of genetically modified cells. A number of improve-ments have occurred in HSC-based gene transfer such thatclinical application is within reach if not already obtained(Cavazzana-Calvo et al., 2000). The inclusion of early-actinghematopoietic growth factors such as Flt-3 ligand and throm-bopoietin and a supportive matrix of either autologous stromaor recombinant fibronectin during transduction has resulted inmarking levels in myeloablated large animals that would be ex-

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FIG. 2. Quantitation of both donor cell engraftment and genemarking in animals conditioned with 800 rads. The top graphshows the engraftment of donor cells measured by flow cy-tometry at the 6-month time point. The solid bars represent themice receiving G1Ne-transduced cells and the open bars rep-resent those receiving the PL-1e-transduced cells. The bottomgraph represents the overall engraftment by genetically modi-fied cells assessed by semiquantitative PCR at 6 months.

FIG. 3. Quantitation of percent donor cell engraftment andgene marking in animals conditioned with 100 rads. (s ) Meanengraftment in mice receiving cells transduced with the neo ex-pression vector; (d ) those receiving cells transduced with thenonexpression vector. The lower panel represents the percent-age of cells positive by semiquantitative PCR analysis in re-cipients of either the neo expression vector transduced cells (s )or the nonexpression vector transduced cells ( d ), assuming onecopy per transduced cell.

pected to be of clinical benefit if achievable in humans (Kiemet al., 1998; Tisdale et al., 1998).

However, not only must gene transfer efficiency to stem cellsbe adequate, but high-level engraftment of these cells must alsooccur. To this end, most strategies have employed high-dose ir-radiation to ablate residual host hematopoietic stem cells andthus remove competition from endogenous nontransduced cells.This then limits the use of such treatments to those patients whocan tolerate the ensuing morbidity as a result of this myeloab-lative conditioning, and for whom the risk–benefit ratio sup-ports the use of such potentially toxic methods. To widen theeventual application of gene therapy approaches to those withnonmalignant disorders, less toxic conditioning is being stud-ied. Apart from the hurdle of overcoming competition fromhigher levels of remaining endogenous cells, the use of lesstoxic conditioning must be adequate to prevent rejection of cellsexpressing foreign transgenes.

Myeloablation has inherent toxicity, and studies have shownit is not required to allow engraftment in the murine setting.Syngeneic stem cells can engraft without prior myeloablation,but very large cell doses were required for engraftment of sex-mismatched murine stem cells at levels of 15–42% 21 to 25months post transplantation (Stewart et al., 1993; Quesenberryet al., 1997). Calculations indicated equal competition on a cellfor cell basis between infused and endogenous stem cells. Thereis data that as little as 100 rads, although not myeloablative andresulting in only minimal depression of peripheral blood counts,may be adequate for high-level engraftment of fresh bone mar-row using clinically achievable cell doses in the mouse model(Mardiney et al., 1997). In our study, we were able to achieveclinically relevant engraftment and levels of genetically modi-fied bone marrow cells after only 100 rads. Without ablation,even very high cell doses of cultured and transduced as opposedto fresh cells have not given quantifiable results due to very

KANG ET AL.1668

FIG. 4. Results of RT PCR for neo transgene expression. PCR product was separated on an agarose gel stained with Sybr green.The upper lanes represent the bands obtained from amplification using neo-specific primer pairs, and the lower lanes using b-actin-specific primer pairs. Lanes labeled with a minus (2) had no reverse transcriptase added. Samples were analyzed from twodifferent mice, labeled 1 and 2, respectively. Both mice were conditioned with 100 rads and received cells transduced with theneo expression vector. The Positive Control lane was obtained from the G1Ne producer cell and the Negative Control, from asample obtained from a nontransplanted mouse.

low engraftment (Schiffmann et al., 1995). Moreover, Peters etal have shown a defect in the engrafting ability of cells culturedand expanded with cytokines (Peters et al., 1995). Even lowerdoses of irradiation may be effective in increasing engraftmentif growth factors are given to the recipient just prior to irradi-ation (Mardiney and Malech, 1996). The number of cells in-fused was chosen to allow easily quantifiable engraftment oftransduced cells, although this cell dose was at least a log higherthan most likely feasible in a large animal or human. Use of exvivo-expanded stem cells may eventually overcome this diffi-culty.

Data from larger animal models suggest that high-dose irra-diation is not required for engraftment of transduced cells. In astudy examining the use of cytokines prior to irradiation, Huhn

et al. demonstrated modest marking levels of 0.1–1.0% after asingle dose of 500 rads in the rhesus macaque, despite usingnow outdated transduction conditions (Huhn et al., 1999).Rosenzweig et al. reported that after conditioning with 400 radsand infusion of transduced peripheral blood stem cells (PBSCs)the level of cells expressing the murine CD24 transgene prod-uct, albeit low, was measurable by flow cytometric analysis outto 4 months. In only 1 of the 4 animals were transgene-reac-tive cytotoxic T lymphocytes (CTLs) found, and their appear-ance did not correlate with the timing of decline in marking asmeasured by PCR in that animal (Rosenzweig et al., 1999). Theconditioning, although not fully myeloablative, was still rela-tively toxic in both studies, with all animals experiencing neu-tropenia, and with one animal from the second study not sur-viving to receive the transplant. Ongoing experiments arestudying very-low-dose irradiation in our own rhesus macaquemodel.

Even with adequate engraftment, cells transduced with a vec-tor expressing a gene perceived as foreign by the recipient maybe cleared by the immune system, and therefore even very hightransduced cell doses may not be adequate for either long-termengraftment and/or disease correction by circulation of cor-rected cells. Mice injected with a vector expressing the humanerythropoietin cDNA developed antibodies to the transgeneproduct. This product was in fact similar enough to the nativemurine erythropoietin such that the antibodies were cross reac-tive, and the mice developed anemia due to an immune responseagainst endogenous murine erythropoietin (Tripathy et al.,1996). Even very little dissimilarity between proteins may per-mit recognition by the immune system such as seen with CD45antigens (van Os et al., 2001). Infusion of transduced lympho-cytes, without any ablation in the rhesus model, resulted in veryrapid clearance of cells containing a neomycin-expressing vec-tor despite long-term persistence of lymphocytes containing anonexpressing vector (Heim et al., 2000). Even in patients withacquired immunodeficiency syndrome (AIDS), in whom thereis an obvious defect of immunity, genetically modified maturelymphocytes are specifically cleared when infused into uncon-ditioned recipients (Riddell et al., 1996). However, in the afore-mentioned rhesus model, following myeloablative conditioningand infusion of transduced HSCs, persistence of the geneticallymodified cells could be demonstrated (Heim et al., 2000).

In solid organ transplantation, microchimerism resultingfrom migration of passenger donor hematopoietic cells can oc-cur (Starzl et al., 1993). There is some evidence that this mi-crochimeric state results in tolerance, and strategies to augmentchimerism have been used to prolong graft survival, generallyby infusing donor bone marrow at the time of organ transplan-tation (Fontes et al., 1994). Attempts have been made to ex-ploit this concept in xenogeneic transplantation as well. Intro-duction of stem cells transduced with retroviral vectorsexpressing either xenoreactive natural antigens (a-Gal) or classI or class II histocompatibility proteins has resulted in slowerrejection rates of allogeneic and xenogeneic grafts, and de-creased antibody formation in both small and large animal mod-els using only minimal or no conditioning radiation or chemo-therapy (Sachs et al., 1993a,b; Sykes et al., 1993; Bracy et al.,1998; Ierino et al., 1999).

Although the results from both solid organ and xenogeneic

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FIG. 5. Quantitation of percent donor cell engraftment asmeasured by flow cytometry and PCR for the GFP transgenein animals conditioned with 100 rads. (s ) Mean engraftmentin mice receiving cells transduced with the GFP expression vec-tor: ( d ) those receiving cells transduced with the nonexpres-sion vector. The lower panel represents the percentage of cellspositive by semiquantitative PCR analysis in recipients of ei-ther the GFP expression vector-transduced cells ( s ) or the non-expression vector transduced cells ( d ).

studies using hematopoietic cells are encouraging, it appearsthat some form of conditioning or immunosuppression may benecessary. In humans, all patients receiving solid organ graftsare treated with immunosuppressants such as cyclosporine toprevent graft rejection. Moreover, in a canine model of alphaID deficiency, unconditioned animals were transplanted withwhole marrow transduced in a long-term culture system, andvector-containing cells disappeared rapidly and in conjunctionwith an immune response against the alpha ID protein, despiteattempts at immunosuppression with cyclosporine post infu-sion. It is possible that the use of their long-term culture sys-tem may have resulted in an infusion of many mature antigen-presenting cells potentially increasing the risk of ananti-transgene immune response (Lutzko et al., 1999).

In our model, we did not study whether similar results couldbe obtained with absolutely no conditioning. The number ofcells required to achieve reproducible and significant levels ofengraftment even using fresh cells without conditioning is notclinically relevant at this time point (Stewart et al., 1993; Que-senberry et al., 1994). Furthermore, as has been shown by oth-ers, as well as from our own unpublished experience, the num-bers must be increased 4- to 100-fold further when usingcultured and transduced cells because the maneuvers requiredto cycle cells and allow transduction appear to result in loss ofengraftment ability (Peters et al., 1995; Dunbar et al., 1998;Stewart et al., 1998). In settings without an intrinsic competi-tive advantage for transduced HSCs or their progeny, even inmouse models, transduced cells compete poorly against en-dogenous stem cells (Quesenberry et al., 1997).

Thus, in our study, we focused on the 100-rad model as mostlikely to eventually be developed clinically. Our results suggestthat only minimal conditioning may be required for introduc-tion of immunogenic transgene products via the hematopoieticstem cell compartment. This very-low-dose irradiation was suf-ficient for engraftment and persistence of genetically modified

cells, but had no discernable toxicity to the mice. Direct evi-dence for persistent in vivo expression of the foreign gene wasdemonstrated using the GFP vector as well as by presence ofneo mRNA in the peripheral blood. Although we have previ-ously shown that expression of the neo gene does not appearto be detrimental to engraftment and or survival of transducedcells, it clearly can be immunogenic (Dunbar et al., 1995; Wuet al., 1998; Heim et al., 2000). Moreover, GFP is even morelikely to be immunogenic in the appropriate setting (Stripeckeet al., 1999). However our results did not show evidence forclearance of the transduced cells. Given that levels of vector-containing cells were similar as assessed by flow cytometry aswell as by PCR, the persistence of transduced cells in our ex-periments is also clearly not the result of gene silencing andtherefore immune escape (Klug et al., 2000).

In conditions in which a selective advantage to the success-fully transduced cells exists and there is a preexisting immu-nodeficient state, no conditioning is necessary, as has beenshown by the recent success obtained in the clinical transplan-tation trial for severe combined immunodeficiency (Cavazzana-Calvo et al., 2000). The ability to achieve high-level, clinicallyrelevant levels of vector-expressing cells using only minimalconditioning for the eventual application of HSC based genetransfer in other nonmalignant disorders where such a selectiveadvantage is not conferred appears feasible, and suggests thatdisappointing results in prior clinical studies have not been re-lated to immune responses against transgene products perceivedas foreign (Malech et al., 1997; Dunbar et al., 1998). These re-sults require confirmation in a large animal model.

ACKNOWLEDGMENT

The authors thank Brian Sorrentino for the MgirL22Yecotropic producer cell line.

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FIG. 6. Representative flow cytometric analysis and quantitation of GFP expression in animals conditioned with 100 rads. Theleft-hand panel shows a representative flow cytometric analysis demonstrating gating used to define GFP-expressing cells. Theright-hand graph represents the mean number of circulating GFP-expressing cells in the entire group of mice over a period of 6months.

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Address reprint requests to:Dr. Cynthia E. Dunbar

Molecular Hematopoiesis SectionHB, NHLBI, NIH

Building 10, Room 7C1039000 Rockville Pike

Bethesda, MD 20892

E-mail: dunbarc@nhlbi.nih.gov

Received for publication February 23, 2001; accepted after re-vision July 19, 2001.

Published online: August 3, 2001.

KANG ET AL.1672